pvuii hf  (New England Biolabs)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans

doi: 10.1073/pnas.1220813110

Figure Lengend Snippet: De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

Article Snippet: Aliquots of 25 μg DNA were digested with 240 U HpaI plus 240 U PvuII-HF (New England BioLabs) in 500 μL NEB4 buffer at 37 °C for 2.5 h. Digested DNA was recovered by ethanol precipitation, dissolved in 5 mM Tris-HCl, pH 7.5, and 15 μg DNA was loaded into a 2.5-cm-wide slot in a 40-cm-long 0.9% (wt/vol) SeaKem HGT (Lonza) agarose gel in 0.5 × tris-borate EDTA buffer containing 0.5 μg/mL ethidium bromide.

Techniques: Mutagenesis, Amplification

Journal: Nature Communications

Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

doi: 10.1038/s41467-021-27004-1

Figure Lengend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

Article Snippet: DNA (1500 ng) was digested using either Bfa I or PvuII-HF (20 U, New England Biolabs) in CutSmart buffer (1×) in 20 µL reactions by incubating at 37 °C for 1 h. Immediately thereafter, reactions were resolved on an agarose gel (1.5%) stained with SYBR Safe (1×), imaged (UVP BioDoc-It), and quantified semi-quantitatively using ImageJ (v1.51j8, raw ImageJ values in Supplementary Data ).

Techniques: Sequencing, Transfection

Journal: Nucleic Acids Research

Article Title: A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

doi: 10.1093/nar/gkab770

Figure Lengend Snippet: High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.

Article Snippet: Using the psoralen-mediated cross-linking conditions indicated in the previous step, around 70–100 units of PvuII-HF enzyme (New England Biolabs, #R3151S) are sufficient to obtain digestion of 10–15 μg of genomic DNA (7–10 units/μg of genomic DNA) in three to five hours at 37°C.

Techniques: In Vivo